An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype.

In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1alpha(1) (DBL1alpha(1)) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1alpha(1) recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1alpha(1)-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1alpha(1) domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

Rosetting is associated with increased Plasmodium falciparum in vivo multiplication rate in the Saimiri sciureus monkey.

Severe Plasmodium falciparum malaria in African children is associated with high peripheral parasite densities and high rate of rosette-forming parasites. To explore the relationship between rosette formation and parasite density in vivo, we compared the multiplication rate of a rosette-forming variant (varO) of the Palo Alto line with a sibling non-rosetting variant (varR) in splenectomized Saimiri monkeys. The multiplication rate of varO parasites was 1.5-fold higher than that of the varR variant. This indicates that rosetting is indeed associated with high parasite multiplication efficiency in vivo and, as such, may contribute to the high parasite densities observed in severe malaria.

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes.

Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

A role for the Plasmodium falciparum RESA protein in resistance against heat shock demonstrated using gene disruption.

During erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3-disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1-KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3-KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 degrees C resulted in 33% culture inhibition of resa1-KO parasites while marginally impacting parental and resa3-KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1-KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.